This proposal is aimed at the identification of the oxygen sensor and the elucidation of the oxygen sensing pathway. Oxygen is essential in many physiological processes, and mechanisms for oxygen sensing seem to exist in all aerobic organisms. Hypoxic inducible factors (HIF) and von Hippel-Lindau (VHL) tumor suppressor proteins are so far the only well-characterized proteins in the mammalian oxygen sensing pathway. Reactive oxygen species generated under normoxia have been shown to trigger the oxidative modification and the subsequent degradation of HIF proteins through VHL-dependent ubiquitylation; consequently, HIF is stabilized only under hypoxic conditions, thereby up-regulating physiologically important target genes, such as erythropoietin and vascular endothelial growth factor. We have cloned and characterized a novel cytochrome b-type NAD(P)H oxidase (b5/b5R) which appears to be a good candidate oxygen sensor. The b5/b5R gene has been identified in human, mouse, rat, fruit flies and nematode worms. The transcript is found in all human cell-lines and tissues. The protein is a NAD(P)H oxidoreductase capable of generating superoxide under normoxic conditions. In this proposal, structural and functional studies will be performed on this candidate in order to elucidate its role in the oxygen sensing and signaling pathway. In Specific Aim 1, mutant b5/b5R proteins with altered binding properties for oxygen and NAD(P)H will be expressed and the kinetics of superoxide production will be measured. Gain- and loss-of-function mutants will be transfected into human cell-lines in order to perturb oxygen sensing. In Specific Aim 2, the structural basis for electron transfer from NAD(P)H through FAD to heme and to oxygen will be studied by both X-ray diffraction analyses and by resonance Raman spectroscopy on wild-type and mutant b5/b5R proteins. In Specific Aim 3, the physical interaction between recombinant HIF, VHL and b5/b5R proteins will be studied in order to reconstitute the subcellular oxygen sensing pathway in vitro. Genetic studies on hypoxic responses in mouse, Drosophila and C. elegans should provide additional functional information on b5/b5R in oxygen sensing and help in the design of the in vitro reconstitution experiments. The knowledge gained from these proposed studies will contribute to our understanding of diverse physiological and pathophysilological processes, including tumorigenesis and adaptation to ischemia.